The present invention relates to the organic synthesis of chemical compounds and anti-cancer pharmaceuticals, particularly macrocyclic lactones having anti-cancer activity and vacuolar ATPase inhibitory activity and methods of synthesis and use of these compounds.
A number of biological metabolites isolated from natural sources such as sponges, tunicates, and bacteria have been found to have anti-cancer activity. These metabolites are macrocyclic lactones such as salicylihalamide and lobatamide, which appear to represent a new mechanistic class based on their cytotoxicity profiles when compared to other compounds in NCI""s standard agents database. Apicularen has also been shown to be cytostatic against human cancer cell lines (Kunze et al. (1998)). Macrolides were previously known to have cytotoxic activity and some possess anti-fungal or anti-bacterial properties. The anti-cancer activity of a class of macrolides called salicylihalamide was discovered by Boyd et al., (1997) in a screen of the cytotoxic activity of extracts from a family of sponges (species Haliclona). Two novel macrolides, salicylihalamides A and B were purified from the extract and tested in the NCI 60-cell line human tumor screen. Upon screening, these compounds demonstrated a mean-graph profile that was unlike any of the known tumor profiles of known anti-tumor agents. Therefore, these compounds represent a new class of anti-tumor agents. These compounds were especially effective against human solid tumor cell lines. Solid tumors are usually the most resistant to drugs.
Various other macrocyclic lactones have been identified as having anti-tumor activity. Included are lobatamides (isolated from Aplidium lobatum), apicularens (isolated from Chondromyces), and oximidines (isolated from Pseudomonas). Lobatamides contain a similar enamide side chain and core structure to salicylihalamides. However, lobatamides contain an oxime methyl ether structure at the end of the enamide side chain. The NCI 60-cell line human tumor screen profile of the lobatamides correlated with the profile of salicylihalamide (McKee et al., (1998)).
Apicularen A causes a potent growth inhibition of human cancer cell lines, the induction of an apoptotic-like cell death, and the formation of mitotic spindles with multiple spindle poles and clusters of bundled actin from the cytoskeleton (Kunze et al., 1998; Jansen et al., 2000).
The NCI 60-cell line human tumor screen is a measure of the effectiveness of a compound for inhibiting or killing various human cancers. It is a set of 60 different cancer cell lines against which chemical compounds can be tested against to determine if the compound has anticancer activity. Each compound has an individual xe2x80x9cfingerprintxe2x80x9d based on effectiveness in killing each of the 60 cancer cell lines.
Firstner et al. (U.S. Pat. No. 5,936,100) has used ring closing metathesis as a step in the synthesis of macrocyclic lactones containing one or more polar functional groups. Macrocycles of ring sizes xe2x89xa712 are challenging to synthesize because the precursors tend to oligomerize.
The naturally occurring structure of salicylihalamide is unstable under certain conditions. Salicylihalamides decompose in CDCl3, due to the unstable side chain (Snider and Song (2000)). The present invention provides macrocyclic lactones which exhibit improved stability over the natural compound. The present invention also includes a process for the synthesis of these compounds which is particularly flexible for making various compounds. The natural compound has not previously been synthesized and its structure was misidentified when it was purified from marine sponges of the genus Haliclona. Boyd et al., in PCT/US98/15011 disclosed the structure of natural salicylihalamide with a negative rotation and assigned the absolute configuration as 12R, 13S, 15R. This assignment was incorrect because the isomer with the 12R, 13S, 15R absolute configuration has a positive rotation and does not have anti-cancer activity, as proven by the inventor in the present application. Only the isomer with the 12S, 13R, 15S absolute configuration has a negative rotation and anti-cancer activity.
The present invention describes the first synthesis of (+)-and (xe2x88x92)-salicylihalamide A and assigns the absolute configuration of the natural product with negative rotation as 12S, 13R, 15S. It a highly efficient, trans-selective ring-closing olefin metathesis for the assembly of the benzolactone skeleton and has been readily adapted to obtain a variety of analogs.
The invention includes a method of synthesis of a broad class of cyclic benzolactones with chemotherapeutic activity which exhibit increased stability over natural benzolactones. Included in the invention are the compounds, compositions containing the compounds, methods of synthesis, and methods of treatment.
An embodiment of the invention is a composition comprising a compound of the formula: 
wherein E is 
X=O, S, NR2; Y=CH2, O, S, NR2;
Q=O, NH;
F=ortho, meta, para substituents such as halogen, CN, OR2, OC(O)R3, NO2, OSO2R3, NR2R2, NR2C(O)R3, NR2SO2R3, R3;
R1=H, Me;
R2=R1, straight chain saturated alkyl, straight chain unsaturated alkyl, branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH2aryl, CH2heteroaryl, CH2heterocycle, CHR1CHR1aryl, CHR1CHR1heteroaryl, CHR1CHR1heterocycle;
R3=R2 or CR1=CR1aryl, CR1=CR1heteroaryl, CR1=CR1heterocycle, Cxe2x89xa1Caryl, Cxe2x89xa1Chetcroaryl, Cxe2x89xa1Cheterocycle; and Z is a contiguous linker whose presence completes an 11 to 15 membered ring. The linker can contain heteroatoms and substituents.
A further embodiment of the invention are compositions comprising compounds of the formulas: 
wherein E is 
X=O, S, NR2 
Y=CH2, O, S, NR2 
Q=O, NH
F=ortho, meta, para substituents such as halogen, CN, OR2, OC(O)R3, NO2, OSO2R3, NR2R2, NR2C(O)R3, NR2SO2R3, R3 
R1=H, Me
R2=R1, straight chain saturated alkyl, straight chain unsaturated alkyl, branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH2aryl, CH2heteroaryl, CH2heterocycle, CHR1CHR1aryl, CHR1CHR1heteroaryl, CHR1CHR1heterocycle R3=R2 or CR1=CR1aryl, CR1=CR1heteroaryl, CR1=CR1heterocycle, Cxe2x89xa1Caryl, Cxe2x89xa1Cheteroaryl, Cxe2x89xa1Cheterocycle; and R4=R1, C(O)R3, SO2R3, R2.
Another embodiment of the invention are compositions comprising compounds of the formulas: 
where F=ortho, meta, para substituents such as halogen, CN, OR2, OC(O)R3, NO2, OSO2R3, NR2R2, NR2C(O)R3, NR2SO2R3, R3;
R1=H, Me;
R2=R1, straight chain saturated alkyl, straight chain unsaturated alkyl, branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH2aryl, CH2heteroaryl, CH2heterocycle, CHR1CHR1aryl, CHR1CHR1heteroaryl, CHR1CHR1heterocycle;
R3=R2 or CR1=CR1aryl, CR1=CR1heteroaryl, CR1=CR1heterocycle, Cxe2x89xa1Caryl, Cxe2x89xa1Cheteroaryl, Cxe2x89xa1Cheterocycle; and
R4=R1, C(O)R3, SO2R3, R2.
An embodiment of the invention is a composition wherein the compound is selected from the group consisting of: 
wherein R=straight chain saturated alkyl or straight chain unsaturated alkyl that is comprised of a chain of 5 to 8 carbons.
An embodiment of the invention is a composition wherein the compound is of the formula: 
wherein R=straight chain saturated alkyl or straight chain unsaturated alkyl that is comprised of a chain of 5 to 8 carbons.
An embodiment of the invention is a composition comprising a compound of the formula: 
An embodiment of the invention is a composition comprising a compound of the formula: 
An embodiment of the invention is a composition comprising a compound of the formula: 
An embodiment of the invention is a composition comprising a compound of the formula: 
An embodiment of the invention is a composition comprising a compound of the formula: 
An embodiment of the invention is a composition comprising a compound of the formula: 
A further embodiment of the invention is the following compositions comprising compounds of the formulas: (These compounds have been tested for growth inhibitory activities against several cell lines, including human melanoma cell line SK-MEL-5 [see Example 7; Table 3], as well a inhibition against reconstituted purified Vacuolar (H+)-ATPase from bovine brain [see Example 7; Table 5]). 
where R=Z,Z-hexadienyl; Z,E-hexadienyl; a straight chain alkyl comprising 5 to 8 carbons (e.g. xe2x80x94(CH2)5Me); a straight chain alcohol (e.g. xe2x80x94O(CH2)4Me); and a straight chain diol (e.g. xe2x80x94S(CH2)4Me); 
where R=Bu; Ph; 
where R=Z,Z-hexadienyl; Z,E-hexadienyl; and a straight chain alkyl comprising 5 to 8 carbons (e.g. xe2x80x94(CH2)5Me); 
where R=a straight chain alkyl comprising 5 to 8 carbons, a straight chain alcohol, a straight chain diol, xe2x80x94CCBu, or xe2x80x94CCph.
Another embodiment of the invention is a composition wherein the compound is selected from the group consisting of: 
wherein R=straight chain saturated alkyl or straight chain unsaturated alkyl that is comprised of a chain of 5 to 8 carbons.
Another embodiment of the invention is a composition wherein the compound is of the formula: 
wherein R=straight chain saturated alkyl or straight chain unsaturated alkyl that is comprised of a chain of 5 to 8 carbons.
Yet another embodiment of the invention is a process for preparing a salicylihalamide comprising the steps of: a) synthesis of a benzolactone core; b) synthesis of an enamide side chain; and c) and addition of the side chain by addition of a dienyllithium (28) to the benzolactone core.
Another embodiment of the invention is a process for preparing a salicylihalamide comprising the steps of: a) synthesis of a benzolactone core; b) synthesis of a side chain; and c) and addition of the side chain to the benzolactone core.
Still another embodiment of the invention is a process for preparing an Apicularen comprising the steps of: a) synthesis of a benzolactone core: b) synthesis of an enamide side chain; and c) and addition of the side chain by addition of a dienyllithium (28) to the benzolactone core.
Another embodiment of the invention is a process for preparing an Apicularen comprising the steps of: a) synthesis of a benzolactone core: b) synthesis of a side chain; and c) and addition of the side chain to the benzolactone core.
Another embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
wherein E is 
X=O, S, NR2; Y=CH2, O, S, NR2;
Q=O, NH;
F=ortho, meta, para substituents such as halogen, CN, OR2, OC(O)R3, NO2, OSO2R3, NR2R2, NR2C(O)R3, NR2SO2R3, R3;
R1=H, Me;
R2=R1, straight chain saturated alkyl, straight chain unsaturated alkyl, branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH2aryl, CH2heteroaryl, CH2heterocycle, CHR1CHR1aryl, CHR1CHR1heteroaryl, CHR1CHR1heterocycle;
R3=R2 or CR1=CR1aryl, CR1=CR1heteroaryl, CR1=CR1heterocycle, Cxe2x89xa1Caryl, Cxe2x89xa1Cheteroaryl, Cxe2x89xa1Cheterocycle; and Z is a contiguous linker whose presence completes an 11 to 15 membered ring. The linker can contain heteroatoms and substituents.
A further embodiment of the invention are compositions comprising compounds of the formulas 
wherein E is 
X=O, S, NR2;
Y=CH2, O, S, NR2;
Q=O, NH;
F=ortho, meta, para substituents such as halogen, CN, OR2, OC(O)R3, NO2, OSO2R3, NR2R2, NR2C(O)R3, NR2SO2R3, R3;
R1=H, Me;
R2=R1, straight chain saturated alkyl, straight chain unsaturated alkyl, branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH2aryl, CH2heteroaryl, CH2heterocycle, CHR1CHR1aryl, CHR1CHR1heteroaryl, CHR1CHR1heterocycle
R3=R2 or CR1=CR1aryl, CR1CR1heteroaryl, CR1=CR1heterocycle, Cxe2x89xa1Caryl, Cxe2x89xa1Cheteroaryl, Cxe2x89xa1Cheterocycle; and
R4=R1, C(O)R3, SO2R3, R2.
Another embodiment of the invention are compositions comprising compounds of the formulas: 
where F=ortho, meta, para substituents such as halogen, CN, OR2, OC(O)R3, NO2, OSO2R3, NR2R2, NR2C(O)R3, NR2SO2R3, R3;
R1=H, Me;
R2=R1, straight chain saturated alkyl, straight chain unsaturated alkyl, branched chain alkyl, branched chain unsaturated alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, CH2aryl, CH2heteroaryl, CH2heterocycle, CHR1CHR1aryl, CHR1CHR1heteroaryl, CHR1CHR1heterocycle;
R3=R2 or CR1=CR1aryl, CR1=CR1heteroaryl, CR1=CR1heterocycle, Cxe2x89xa1Caryl, Cxe2x89xa1Cheteroaryl, Cxe2x89xa1Cheterocycle; and
R4=R1, C(O)R3, SO2R3, R2; 
where R=Z,Z-hexadienyl; Z,E-hexadienyl; a straight chain alkyl comprising 5 to 8 carbons, a straight chain alcohol or a straight chain diol; 
where R=Bu; Ph; 
where R=Z,Z-hexadienyl; and a straight chain alkyl comprising 5 to 8 carbons; 
where R=a straight chain alkyl comprising 5 to 8 carbons (e.g. xe2x80x94(CH2)5Me), a straight chain alcohol (e.g. xe2x80x94O(CH2)4Me), a straight chain diol (e.g. xe2x80x94S(CH2)4Me), xe2x80x94CCBu, or xe2x80x94CCph.
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula selected from the group consisting of: 
wherein R=straight chain saturated alkyl or straight chain unsaturated alkyl that is comprised of a chain of 5 to 8 carbons.
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula selected from the group consisting of: 
wherein R=straight chain saturated alkyl or straight chain unsaturated alkyl that is comprised of a chain of 5 to 8 carbons.
An embodiment of the invention is a method of treating or preventing cancer, comprising the step of administering to a patient a therapeutically effective amount of a compound of the formula: 
wherein R=straight chain saturated alkyl or straight chain unsaturated alkyl that is comprised of a chain of 5 to 8 carbons.
An embodiment of the invention is a method for treating cancer comprising the step of contacting a tumor cell within a subject with a macrocyclic lactone of the present invention under conditions permitting the uptake of said macrocyclic lactone by said tumor cell. The tumor cell may be derived from a tissue selected from the group consisting of brain, lung, liver, spicen, kidney, lymph node, small intestine, blood, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head, and neck, esophagus, and bone marrow. In a further embodiment, the subject is human. In another embodiment, the macrocyclic lactone is contained within a liposome. In yet another embodiment, the macrocyclic lactone is administered intratumorally, in the tumor vasculature, local to the tumor, regional to the tumor, or systemically. In a further embodiment, the method comprises administering a second chemotherapuetic agent to said subject. In a further embodiment, the second chemotherapeutic agent may be cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, famesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate or any analog or derivative variant of the foregoing. In another embodiment, the method further comprises administering radiation to said subject. In another embodiment, the radiation is delivered local to a cancer site. In yet another embodiment, the radiation is whole body radiation. The radiation may be xcex3-rays, X-rays, accelerated protons, microwave radiation, UV radiation or the directed delivery of radioisotopes to tumor cells. In another embodiment, the method further comprises administering an anticancer gene to said subject. In an embodiment of the invention, the anticancer gene is a tumor suppressor. In another embodiment of the invention, the anticancer gene is an inhibitor of apoptosis. In another embodiment of the invention, the anticancer gene is an oncogene antisense construct.
It is a further embodiment of the present invention to provide a method for inhibiting vacuolar ATPase (V-ATPase) proton-pumping activity. The method comprises contacting V-ATPase with the compounds of the present invention in an amount sufficient to inhibit the ATPase proton-pumping activity of the V-ATPase. Inhibition of the V-ATPase proton pumping activity by the compounds of the present invention is useful, inter alia, for the treatment and prevention of cancer and osteoporosis.
An embodiment of the invention is a method for altering the phenotype of a tumor cell comprising the step of contacting the cell with a macrocyclic lactone of the present invention, under conditions permitting the uptake of said macrocyclic lactone by said tumor cell. The tumor cell may be derived from a tissue selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, blood, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head, and neck, esophagus, and bone marrow. In an embodiment of the invention, the phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth, cell cycling, invasiveness, tumorigenesis, and metastatic potential. In another embodiment of the invention, the macrocyclic lactone may be contained within a liposome.
Another embodiment of the invention is a method of treating a subject with cancer comprising the step of administering to said subject a macrocyclic lactone of the present invention under conditions permitting the uptake of said macrocyclic lactone by said cancer cell. In an embodiment of the invention, the subject is human.
Another embodiment of the invention is a method of suppressing growth of a tumor cell comprising contacting said cell with a macrocyclic lactone of the present invention, under conditions permitting the uptake of said macrocyclic lactone by said tumor cell. In another embodiment, the macrocyclic lactone is contained within a liposome.
Another embodiment of the invention is a method of regulating cell growth and proliferation in normal and malignant cells, comprising the step of administering, to an individual in need of said treatment, a therapeutically effective amount of a compound of the present invention.
Another embodiment of the present invention is a method of inhibiting growth of proliferating cells comprising the step of administering, to the proliferating cells, a therapeutically effective amount of a compound of the present invention.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.